The principal goals of the High Throughput Screening Core at UT Southwestern Medical Center (UTSW) are to aid in the early, pre-clinical discovery and development of new small molecule therapeutics and to support the identification and characterization of novel biological targets and pathways for therapeutic intervention in areas of unmet medical need. The first goal is addressed through full-file and subset screening of our compound library (~330,000 compounds) and by supporting confirmation, dose-response studies, and secondary activity profiling of selected hit compounds as well as synthesized and purchased synthetic analogs. In the UT Southwestern FusOnC2 U54 Center, we will work with Project 3 to optimize secondary and counter screen assays and provide follow-up support for confirmed hits from the 8,000 compound pilot screen. We will execute and analyze a high content screen against the UT Southwestern Chemical Library (~330,000 drug-like small molecules), structurally cluster and annotate the hits in the screen (absolute Z-scores > 3) using historical data and substructure alerts, and facilitate hit picking for confirmation studies for Project 3 and Medicinal Chemistry Core. We will carry out primary hit confirmation studies and secondary assay profiling of 1,900 primary hits from the 300K primary screen in a dose-response format with Project 3 to enable hit selection for medicinal chemistry follow-up. In collaboration with Project 2, we will develop a robust, 384 well-compatible high throughput assay (Z' > 0.5) for measuring the modulation of the stability of cross-beta polymers formed by low complexity sequences of FET Proteins (FUS, EWSR1-FLI1, TAF-15). We will optimize secondary and counter screen assays and provide follow-up support for confirmed hits from the 8,000 compound pilot screen. As described above in Aim 2, we will support the large-scale screen of our institutional library for Project 2 and work collaboratively with Project 2 and the Medicinal Chemistry and Pre-clinical Pharmacology Cores in optimizing hits from this screen into potential leads for the treatment of Ewing sarcoma. Aim 3 is to provide access to and support for functional genomic screens using the arrayed, genome-wide CRISPR library in Project 1.